Synthesis of lactulose via semi-rational design Aided evolution of glucose isomerase

Directed evolution of the active pocket is particularly effective for altering the substrate specificity of enzymes. Herein, we investigate the feasibility of lactulose synthesis by glucose isomerase based on re-sharping the active pocket. The glucose isomerase from Streptomyces sp. SK ( SK GI) was found possessing the activity to isomerize lactose. Based on this discovery, directed evolution of SK GI was performed via re-construction of the substrate binding pocket to improve its substrate specificity of towards lactose. As a result, the variant SK GI (N215C/K289R) with higher production (3.33-fold improvement) and longer half-life (increased from 196.92 min to 278.37 min) was obtained. These findings will provide new research perspectives for the directed evolution of enzymes. In the future, they will drive enzymes to evolve new functions by overcoming catalytic limitations.